An important enzyme involved in one carbon metabolism is the NADP-dependent trifunctional enzyme MTHFD1 (5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase, 10-formyltetrahydrofolate synthetase) [Hum et al., J. Biol. Chem., 263:15946-15950 (1988)]. MTHFD1 is often referred to as the “C1-THF synthase” and catalyses the interconversion of tetrahydrofolate to 10-formyl, 5,10-methenyl, and 5,10-methylene derivatives. These derivatives form an important part of de novo DNA synthesis. Promotion of DNA synthesis is desirable in placental and fetal development. In other contexts, such as cancer treatment, blocking of DNA synthesis is desirable. Maternal folate status and/or homocysteine levels have been implicated in a range of pregnancy-related complications, most notably in pregnancies affected by a neural tube defect (NTD).
A polymorphic variant at position 1958 of MTHFD1 at which guanine is replaced with an adenosine results in the substitution of a conserved arginine amino acid with a glutamine at position 653. One study has disclosed that this polymorphic variant is a maternal risk factor for neural tube defects (NTDs) [Brody et al., Am. J. Hum. Genet., 71:1207-1215 (2002)]. Neural tube defects (NTDs) are common congenital malformations that can be presented as anencephaly, encephalocele, and spina bifida. NTDs' etiology likely includes both genetic and environmental factors. Intervention trials have shown that maternal supplementation with folic acid in the period before pregnancy can prevent the majority of NTD-affected pregnancies.
Abruptio placentae or placental abruption is thought to arise from a sudden rupture of the spiral arteries, resulting in the premature separation of a normally implanted placenta [Anath et al., Obstet. Gynecol. 88:309-318 (1996); Eskes, Eur. J. Obstet. Gyn. R. B. 95:206-212 (2001)]. This event leads to increased risk of adverse outcomes to both mother and baby. The underlying cause of abruptio placentae is unknown, but several factors have been suggested to increase risk including folate deficiency, hyperhomocysteinemia, preeclampsia and history of a prior pregnancy abruption [Kramer et al., Obstet. Gynecol. 89: 221-226 (1997); Misra et al., J. Clin. Epidemiol. 52: 453-461 (1999); Ray et al., Placenta, 20: 519-529 (1999); Eskes, Eur. J. Obstet. Gyn. R. B., 95: 206-212 (2001).] Non-genetic risk factors have been described including cigarette smoldng, preeclampsia and increased maternal age [Misra et al., J. Clin. Epidemiol. 52: 453-461 (1999); Eskes, Eur. J. Obstet. Gyn. R. B. 95: 206-212 (2001)]. Additional risk factors include elevated homocysteine [Goddijn-Wessel et al., Br Med. J., 2: 1431-1436 (1996); van der Molen, et al., Am. J. Obstet. Gynecol., 182: 1258-1263 (2000)] and low folate levels [Hibbard et al., Br. Med. J., 2: 1431-1436 (1963); Streiff et al., N. Engl. J. Med., 276: 776-779 (1967); Whalley et al., Am. J. Obstet. Gynecol., 105: 670-678 (1969); Hibbard, S Afr Med J 49: 1223-1226 (1975); Goddijn-Wessel et al., Br. Med. J., 2: 1431-1436 (1996)].
A substantial proportion (15-50%) of second trimester pregnancy losses remain unexplained [Gaillard et al., Arch. Pathol. Lab. Med, 117:1022-1026 (1993); Faye-Petersen et al., Obstet. Gynecol. 94, 915-920 (1999); Incerpi et al., Am J Obstet Gynecol 178, 1121-1125 (1998); Drakeley et al., Hum Reprod 13, 1975-1980 (1998)]. Although placental insufficiency is a common finding in these cases {Faye-Petersen et al., Obstet. Gynecol. 94, 915-920 (1999)], its etiology is often unknown. Sub-optimal folate or B12 metabolism due to either a deficient diet or a genetic predisposition appears to increase the risk of a number of pregnancy complications including spontaneous abortion.
Polymorphisms have been studied in the context of a variety of cancers and other diseases. [See, e.g., Chen, et al., Int. J. Cancer, 110, 617-620 (2004), Krajinovic et al., The Pharmacogenomics Journal 4:66-72 (2004); U.S. Pat. Nos. 5,449,605; 5,688,647; 5,719,026; 5,942,390; 6,294,399; 6,312,898; 6,537,759; 6,548,245; 6,627,401; 6,664,062; 6,759,200; 6,818,758; 6,833,243, and 6,872,533; and U.S. Patent Application Publication Nos: 2005/0084849; 2005/0089905; 2005/0095593; and 2005/0112680.
Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) is a trifunctional enzyme localized to mitochondria which has been reported to have one or more enzymatic activities in common with MTHFD1. MTHFD1L has been shown to be transcribed into two mRNA transcripts: 1.1 kb and 3.6 kb in size. The shorter transcript is produced by splicing Exon7 with alternative Exon8A and therefore it lacks the 10-formyltetrahydrofolate synthase (synthetase) sequence [Prasannan et al., J. Biol. Chem., 278(44):43178-43187 (2003)].
Methylenetetrahydrofolate reductase (MTHFR) is involved in the remethylation of homocysteine to methionine by generating the necessary methyl group donor 5-methyltetrahydrofolate from 5,10-methylenetetrahydrofolate. Polymorphisms within MTHFR have been extensively studied in relation to a wide variety of diseases including NTDs, cancer and pre-eclampsia. The MTHFR polymorphic variant at 677 where a C is replaced with a T has a functional effect on the MTHFR enzyme and is associated with elevated plasma homocysteine levels when folate status is low. A relatively small number of studies have examined the MTHFR polymorphisms in relation to abruptio placentae and results have not yielded a clear indication of increased risk for the disorder.
Coagulation factor II is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss. F2 also plays a role in maintaining vascular integrity during development and postnatal life. Mutations in F2 can lead to various forms of thrombosis and dysprothrombinemia.
Coagulation factor V (F5) is an essential factor of the blood coagulation cascade, and circulates in plasma, and is converted to the active form by the release of the activation peptide by thrombin during coagulation. Once activated, factor V is a cofactor that participates with activated coagulation factor X to activate prothrombin to thrombin. Defects in this gene result in either an autosomal recessive hemorrhagic diathesis or an autosomal dominant form of thrombophilia, which is known as activated protein C (APC) resistance. A variant of factor V with a particular single point mutation associated with APC resistance is known as factor V Leiden [Bertina et al., Nature, 369:64-67 (1994)].
Transcobalamin II (TCNII) is a member of the vitamin B12-binding protein family. TCNII binds cobalamin and mediates the transport of cobalamin into cells. TCNII polymorphic variant 776C>G (P259R) has been reported to confer an increased fetal genetic risk of early spontaneous abortion [Zetterberg et al., Hum. Reprod., 17:3033-3036 (2002)] and influence levels of circulating vitamin B12 bound to TCNII [Afman et al., Eur. J. Hum. Genet., 10:433-438 (2002), Miller et al., Blood, 100:718-720 (2002)]. TCNII 776C>G polymorphic variant may interact with the MTHFR 677TT genotype to confer an even higher fetal genetic risk of spontaneous abortion than either polymorphism separately [Zetterberg et al., Hum. Reprod., 18:1948-1950 (2003)].
Chemotherapy of cancer has involved use of highly toxic drugs with narrow therapeutic indices, and, most adult solid cancers remain highly resistant to treatment. Chemotherapy often results in a significant fraction of treated patients suffering unpleasant or life-threatening side effects while receiving little or no clinical benefit; other patients may suffer few side effects and/or have complete remission or even cure. Chemotherapy is also expensive, based not only on the cost of drugs, but the medical care involved with their administration. Tests are needed that better predict chemotherapy efficacy; such tests would allow for more selective use of toxic drugs. In those cases where toxicity of chemotherapy or other drug regemin is at least partially a result of genetic differences, the identification of relevant polymorphic variants will allow for more effective and safer drug use.
Accordingly, to better diagnose and treat pregnancy complications and neoplastic disorders, cardiovascular disorders, Alzheimer's disease and other conditions associated with one carbon metabolic patheways, there is a need to identify polymorphic variants that indicate a relative susceptibility to such diseases and/or relative response to treatments for such diseases.